Preparing and Injecting Embryos of Culex Mosquitoes to Generate Null Mutations using CRISPR/Cas9

Summary

CRISPR/Cas9 is increasingly used to characterize gene function in non-model organisms. This protocol describes how to generate knock-out lines of Culex pipiens, from preparing injection mixes, to obtaining and injecting mosquito embryos, as well as how to rear, cross, and screen injected mosquitoes and their progeny for desired mutations.

Abstract

Culex mosquitoes are the major vectors of several diseases that negatively impact human and animal health including West Nile virus and diseases caused by filarial nematodes such as canine heartworm and elephantasis. Recently, CRISPR/Cas9 genome editing has been used to induce site-directed mutations by injecting a Cas9 protein that has been complexed with a guide RNA (gRNA) into freshly laid embryos of several insect species, including mosquitoes that belong to the genera Anopheles and Aedes. Manipulating and injecting Culex mosquitoes is slightly more difficult as these mosquitoes lay their eggs upright in rafts rather than individually like other species of mosquitoes. Here we describe how to design gRNAs, complex them with Cas9 protein, induce female mosquitoes of Culex pipiens to lay eggs, and how to prepare and inject newly laid embryos for microinjection with Cas9/gRNA. We also describe how to rear and screen injected mosquitoes for the desired mutation. The representative results demonstrate that this technique can be used to induce site-directed mutations in the genome of Culex mosquitoes and, with slight modifications, can be used to generate null-mutants in other mosquito species as well. 

Introduction

Culex mosquitoes are distributed throughout the temperate and tropical regions of the world and transmit several deadly viruses including West Nile virus¹, St. Louis encephalitis² as well as filarial nematodes that cause canine heartworm³ and elephantiasis⁴. Members of the Culex pipiens complex, which includes Cx. quinquefasciatus, Cx. pipiens pipiens and Cx. pipiens molestus, show striking variations in many aspects of their biology. For example, while Cx. quinquefasciatus and Cx. pipiens molestus are ....

Protocol

In most research institutions, an approved Biosafety Protocol must be in place before transgenic insects are generated or maintained to ensure that genetically modified organisms will not escape or be removed from the laboratory facility. Additional government regulations might also apply. Before beginning a project of this nature, check all institutional policies and procedures to determine what documents and approvals are required.

1. Designing gRNAs and preparing injection mixes

1. Design 2-5 gRNAs for each target gene as some gRNAs work better than others. gRNAs that target a gene of interest can be either manu....

Results

Using the described protocol, we were able to successfully inject embryos of Cx. pipiens, and observed a high rate of survival among the injected embryos (~55%, Figure 1). Earlier trials had a lower percentage of survival, likely because the anterior of the egg follicle was attached to the medical dressing strip, preventing mosquito larvae from escaping from the chorion and successfully swimming into the water. Ensuring that the anterior end extends beyond the strip of medical dress.......

Discussion

This protocol presents methods to introduce specific mutations into the genome of Culex mosquitoes and can be used to edit the genome of other mosquitoes as well. The protocol is significant in that it provides specific details of not only how to prepare the injection materials, but also a detailed video overview of how to induce mosquitoes to lay eggs, as well as how to prepare and inject those eggs. We also summarize of how to take advantage of the biology of female Cx. pipiens to lay eggs in individu.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Artificial Membrane Feeder	Hemotek	SP5W1-3	Company location: Blackburn, UK
ATP	Invitrogen	18330019	Company location: Carlsbad, CA, USA
Borosilicate glass mirocapillary tubes, 1 mm outer diameter	World Precision Instruments	1B100-6	Company Location: Sarasota, FL, USA
BV10 Needle Beveler	Sutter Instruments	BV-10-B	Company Location: Nobato, CA, USA
Whatman Circular filter paper (12.5 cm)	Sigma Aldrich	WHA1001125	Company Location: St. Louis, MO, USA
Conical tube (50 mL)	Thermo Fisher Scientific	339652	Company Location: Waltham, MA, USA
Fisherbrand course filter paper with fast flow rate	Thermo Fisher Scientific	09-800	Company Location: Waltham, MA, USA
Cover glass (24 x 40 mm)	Thermo Fisher Scientific	50-311-20	Company Location: Waltham, MA, USA
Dental dam	Henry Schein Inc	1010171	Company Location: Melville, NY USA
Scotch double-sided tape	Thermo Fisher Scientific	NC0879005	Company Location: Waltham, MA, USA
FemtoJet 4i microinjector	Eppendorf	5252000021	Company Location: Hamburg, Germany
Glass vial (2 dram)	Thermo Fisher Scientific	033401C	Company Location: Waltham, MA, USA
Halocarbon oil	Sigma Aldrich	H8898-50ML	Company Location: St. Louis, MO, USA
P-2000 Laser Needle Puller	Sutter Instruments	P-2000/G	Company Location: Nobato, CA, USA
Parafilm	Thermo Fisher Scientific	50-998-944	Company Location: Waltham, MA, USA
PC-100 Weighted Needle Puller	Narishige	PC-100	This is compatabile with the earlier PC-10 model, which has been discontinued. Company Location: Amityville, NY, USA
Phire Direct PCR Kit	Thermo Fisher Scientific	F140WH	Company Location: Waltham, MA, USA
Kodak Photo-Flo (1%)	Thermo Fisher Scientific	50-268-05	Company Location: Waltham, MA, USA
Quartz glass mirocapillary tubes, 1 mm outer diameter	Capillary Tube Supplies Limited	QGCT 1.0	Company Location: Cornwall, UK
Guide-it™ sgRNA Screening Kit	Takara, Bio USA	632639	This kit allows you to determine if gRNAs cut DNA sequences in vitro. Company Location: Mountain View, CA, USA
Sigmacote	Sigma Aldrich	SL2-100ML	Company Location: St. Louis, MO, USA
Small petri dishes (35X10 mm)	Thermo Fisher Scientific	50-190-0273	Company Location: Waltham, MA, USA
Sodium citrate chicken blood	Lampire biologicals	7201406	Company Location: Everett, PA, USA
Fisherbrand Square petri dish (10 cm x 10 cm)	Thermo Fisher Scientific	FB0875711A	Company Location: Waltham, MA, USA
Tegaderm	Henry Schein Inc.	7771180	Company Location: Melville, NY USA
Tropical fish food	Tetramin	N/A
Whatman filter paper	Thermo Fisher Scientific	09-927-826	Company Location: Waltham, MA, USA
Whatman filter paper, 4.25 cm	Sigma Aldrich	1001-042	Company Location: St. Louis, MO, USA